This proposal will examine the role of netrins and semaphorins for axon guidance in the zebrafish. The discovery of semaphorins and netrins has lead to an appreciation of chemoattractive and repulsive mechanisms for axon guidance. However, our understanding of the in vivo action of these molecules is still incomplete. 1. The semaphorins are a large family of molecules, but aspects of their function is only known for 3 semaphorins. This proposal will examine the in vivo action of 2 more semaphorins. 2. It is clear that several mechanisms work coordinately to guide axons. We now have some understanding of how individual netrins and semaphorins work, but do not yet know how these molecules work together to guide specific growth cones. This proposal will see how these molecules work in concert to guide spinal commissural axons. 3. Commissural growth cones first extend toward the midline and then away from the midline in the CNS. How they do this despite the presence of bilaterally symmetric guidance cues, such as chemoattractive netrins expressed at the ventral midline of the CNS, is not well understood. This proposal tests a mechanism that accounts for this behavior. Based upon our analysis of netrins and semaphorins in zebrafish and the actions of these molecules in other organisms, a model that incorporates 2 netrins and 2 semaphorins was generated to account for pathfinding by spinal commissural axons. The critical feature of the model is that commissural growth cones change their responses to these molecules at the ventral midline, perhaps as a result of interactions with the floor plate. Commissural growth cones extend ventrally on the ipsilateral side of the cord because they are repulsed by Sema Z2 secreted by the roof plate and attracted by Netrin-1a and Netrin-1b secreted by the ventral half of the cord and floor plate, respectively; pause at the floor plate to reprogram themselves; and then extend ventrally on the contralateral side because they are insensitive to or attracted by Sema Z2, insensitive to or repulsed by Netrin-1b, and insensitive to or remain attracted to Netrin-1a. Furthermore, Sema Z7 which is now expressed by the dorsal cord repulses the growth cones so that they now turn to extend longitudinally in the dorsal cord. Our basic strategy will be to manipulate semaphorins and netrins and determine how this affects the behavior of growth cones in zebrafish embryos. In addition, we will test the redundancy proposed in the model by examining commissural growth cones in spinal cords missing both the roof and floor plate so that they are devoid of molecules including Sema Z2 and Netrin-1b derived from the roof and floor plate.